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U.S. Chamber of Commerce President and CEO Thomas Donohue had much to celebrate when the business lobby marked its centennial last weekend. He also had cause for alarm.

The chamber has unleashed its costliest, most ambitious political mobilization effort ever, targeting about a dozen Senate and more than 50 House races in a campaign that, by some estimates, will top $50 million. It’s also spent more than $86 million on lobbying since the beginning of last year, outpacing all other advocacy groups.

But the chamber, which has been trumpeting its milestone in videos on YouTube and elsewhere, could be facing the political fight of its life. The Republican Party’s shift to the right has put the group at odds with lawmakers and advocacy groups allied with the tea party, fueling intraparty rifts on Capitol Hill and on the campaign trail. In addition, a new generation of super PACs that can raise and spend unlimited money is crowding the airwaves in key races across the country, threatening to drown out the chamber’s message.

The organization’s increasingly aggressive lobbying and advocacy stance has also riled erstwhile allies, including local chambers of commerce, small-business advocates and corporations such as Yahoo and PG&E Corp. And the group faces pressure from consumer and shareholder advocates that oppose undisclosed political spending. As a 501(c)(6) trade group, the chamber operates outside campaign disclosure rules.

The recent progressive campaign to pressure more than a dozen corporations — including Coca-Cola, Kraft Foods and Mars Inc. — to withdraw from the conservative American Legislative Exchange Council could serve as “a model that can be replicated” to encourage “similar defections from the chamber,” Christy Setzer, a spokeswoman for U.S. Chamber Watch, a labor-backed watchdog group, said in an email.

“There are always people who are going to oppose the business community,” responded chamber Senior Vice President Rob Engstrom, the group’s national political director. “We welcome the debate every single time.”

Engstrom said friction with some House Republicans elected with the chamber’s help in 2010 has been overblown. Several recent chamber-backed measures — including legislation to raise the debt limit, reauthorize highway programs and renew the charter of the U.S. Export-Import Bank — have run aground amid conservative opposition.